GSM6509111: scRNA_Mouse_ILC_Spleen and Meninges; Mus musculus; RNA-Seq
The meninges (50 mice divided into three samples) and spleens (two mice per sample) of C57BL/6 mice were enzymatically digested in DMEM collagenase 1mg/ml for 30 min at 37°C. Digestion reaction was stopped by addition of DMEM 10% FBS and dissociation into single cell suspension is finalized by mechanical disruption on 70 μm cell strainer (Falcon, Beckton Dickinson). The cell suspensions were washed and resuspended in staining buffer (PBS without Ca2+ Mg2+ 205 , BSA 0,5%, EDTA 2mM and NaN3 0,025%), tagged with BD single cell multiplexing kit, mixed and stained with Zombie Violet for 15 min at room temperature for exclusion of dead cells and anti-CD16/32 blocking mAb (clone 24G2) for 10 min at 4°C followed by incubation  with anti-CD45, -CD3, -NK1.1 mAbs for 25 min. CD45+CD3- NK1.1+ gated cells were sorted into PBS buffer 2% BSA 0,5mM EDTA using a FACSAriaIII (BD Biosciences) equipped with a 488, 561 210 and a 633nm laser and FACSDiva software (BD Biosciences version 6.1.3), and retained on ice. To reduce stress, cells were isolated in gentle FACS-sorting conditions using a ceramic nozzle of size 100μm, a low sheath pressure of 19.84 pound-force per square inch (psi) that maintain the sample pressure at 18.96 psi and an acquisition rate of maximum 1500 events/s. RNA was extracted according to the manufacter's instructions (BD Rhapsody™ Single-Cell Analysis System ). Library was performed according to the manufacter's instructions (BD Rhapsody™ Single-Cell Analysis System ).